The fusion of myeloma cells to spleen cells from immunized mice by Kohler and Milstein in 1975 [Nature 256 495-597 (1975)] demonstrated for the first time that it was possible to obtain a continuous cell line making homogenous (so called "monoclonal") antibodies. Since this seminal work, much effort has been directed to the production of various hybrid cells (called "hybridomas") and to the use of the antibody made by these hybridomas for various scientific investigations.
The general method of production of hybrid cell lines of the type described above comprises the steps of immunizing a animal (usually a rat or mouse, but not necessarily one of these) with an antigen to which a monoclonal antibody is required. After allowing time for the immune system to generate lymphocytes secreting the antibodies to the antigen, the animal is sacrificed and a suspension of spleen cells is prepared. Fusion between these cells and myeloma cells is achieved by bringing them into contact in the presence of a fusion promoter (e.g. polyethylene glycol). A small percentage of the cells fuse to produce hybrid myeloma cells. The immunization results in a plurality of different lymphocytes each secreting antibody to a different antigenic determinant, and these characteristics are transferred genetically to the hybrid cells. It is possible, by careful screening, to isolate from a culture of hybrid cells, a cell having the desired specificity. Such cells may be cloned and cultured.
The advantage of this technique is that it provides a source of a specific antibody uncontaminated by antibodies raised to other determinants either on the antigen with which the mammal was immunized or on antigenic impurities in the immunizing material. Another advantage of the technique is that antigen not available in a pure form for screening assays and present in the immunizing material at low concentrations, may be used.
The present invention concerns a monoclonal antibody to bronchopulmonary carcinomas that may classify various bronchopulmonary carcinomas and identify one specific lung carcinoma type, adenocarcinoma, while additionally identifying adenocarcinoma in non-pulmonary sites.
The increasing clinical and epidemiologic significance of bronchopulmonary carcinomas is a matter of serious concern. Pathologically, bronchopulmonary neoplasms encompass several types: squamous cell carcinoma (SCC), adenocarcinoma (AC), adenosquamous carcinoma (ASC), large cell carcinoma (LCC), bronchioloalveolar carcinoma (BAC), small cell neuroendocrine carcinoma (SCNC), intermediate cell neuroendocrine carcinoma (ICNC), well-differentiated neuroendocrine carcinoma (WDNC), carcinoid, and other rare entities such as mucoepidermoid carcinoma, adenoid cystic carcinoma and carcinosarcoma.
The classification of bronchopulmonary neoplasms on the basis of conventional morphology is difficult, because the pattern of differentiation of individual neoplasms may vary markedly; and, the diagnostic criteria may vary considerably among pathologists. Moreover, one may see carcinomas with various admixtures of SCC, AC, and neuroendocrine (NE) carcinoma differentiation, etc. There are also "poorly" "differentiating carcinomas", which by conventional light microscopy do not express a definite pattern of differentiation; these are usually classified at LCC's. For purposes of this invention, bronchopulmonary refers to the bronchial tubes, pulmonary system and lungs.
The terminology used in identifying carcinomas is often difficult to interpret because of the rapidly changing art being developed to classify carcinomas. As more characteristics of carcinomas are discovered, they may be re-classified into different groups.
It would be most helpful to pathologists to have monoclonal antibodies available for the classification of the different types of bronchopulmonary carcinomas and one that would specifically identify adenocarcinoma. Additionally, it would be helpful to pathologists if the monoclonal antibody will be reactive in fixed paraffin embedded tissues. Many antibody preparations are only useful in flow cytometric analysis, with frozen section, and in radial immuno- and ELISA assays. It is more convenient to use standard immunohistochemical techniques to study the phenotypic characteristics of tissues and tumors. In particular, there is a recognized need for monoclonal antibodies which may classify bronchopulmonary carcinomas and specifically identify bronchocarcinomas and adenocarcinomas throughout the body and which may be used with fixed paraffin embedded tissues.
Several investigators have attempted to produce monoclonal antibodies that will distinguish the various types of lung carcinomas. Brenner et al. (Brenner B. G., Jothy S., and Shuster J. Monoclonal Antibodies to Human Lung Tumor Antigens Demonstrative by Immunofluorescence and Immunoprecipitation Cancer Research 42:3187-3192 (1982) describe the production of monoclonal antibodies directed against glycoprotein antigens with molecular weights of 25,000 KD and 11,500 KD that had been purified from a squamous cell lung cancer extract by anti-B.sub.2 -microglobulin affinity chromatography. Varki et al. (Varki N. M., Reisfield R. A., Walker L. E. Antigens Associated with Human Lung Adenocarcinoma defined by Monoclonal Antibodies. Cancer Research 44:681-687, (1984) discuss three monoclonal antibodies raised against UCLA P.sub.3, an adenocarcinoma of the lung cell line; K S 1/4 and K S 1/17 recognizing different epitopes of glycoprotein with a molecular weight of 40,000 KD and KS 1/9, recognizing a presumed glycolipid. With these antibodies, cross reactivity was seen with all types of lung carcinomas. Mazauric et al. (Mazauric T., Mitchell K. F., Letchworth G. J. III, et al. Monoclonal Antibody-Defined Human Lung Cell Surface Protein Antigen. Cancer Research 42:150-154 (1982) produced four monoclonal reagents against a variety of human lung carcinoma cell lines whose provenance was not clearly detailed. The antibodies reacted with glycoproteins with a molecular weight of 37,000 and 19,000; 127,000; 126,000; 149,000; and 119,000. A limited number of tumor cell lines were tested to establish their binding profile. Brown, et al. (Brown D. T. and Moore M.) Monoclonal Antibodies Against Two Human Lung Carcinoma Cell Lines. Br. J. Cancer 46:794-801, (1982) isolated two monoclonal antibodies recognizing antigens present in squamous cell lung carcinoma. The antigens were quantatively abundant in squamous cell carcinomas but were not restricted to this tumor type. Identification of the target antigens was not accomplished. Mulshine et al. (Mulshine J. et al., Cuttitta F., Bibro M., et al.) Monoclonal Antibodies that Distinguish Non-Small Cell Lung Cancer from Small Cell Lung Cancer. J. Immuno 131:497-502, (1983) have reported two monoclonal antibodies directed against different epitopes of a 31,000 KD protein present in the cytoplasm of large cell lung carcinoma. These antibodies bound to 11/13 non-SCLC cell lines and 0/11 SCLC cell lines. The antigen was present in several non-lung cancer tumors. In addition, Rosen et al. (Rosen S. T., Mulshine J. L., Kuti F., et al.) Analysis of Human Small Cell Lung Cancer Differentiation Antigens Using a Panel of Rat Monoclonal Antibodies. Cancer Research 44:2052-2061, (1984) have poduced a panel of rat IgM monoclonal antibodies that recognize glycolipid antigens associated with several types of pulmonary carcinomas.